Page 137 - Haematologica April 2020

Clofazimine inhibits chronic myeloid leukemia
We next evaluated whether the introduction of exoge- nous p65 could affect clofazimine’s actions and found that p65 overexpression did indeed ameliorate the clofaz- imine-mediated decrease in MYB and PRDX1 expression, increased caspase-3 cleavage (Figure 5H), apoptosis, differ-
entiation and ROS production in K562 cells (Figure 5I-K, Online Supplementary Figure S9A, B). There was a trend (albeit not statistically significant) to increased p65 mRNA in CP-CML cells compared to the level in cells from healthy donors (Figure 5L). Together, the results indicate
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Figure 5. Clofazimine regulates myeloblastoma oncoprotein expression via rapid proteasomal degradation of p65 NFκB. (A) Transfected p65 or tumor necrosis fac- tor-α (10 ng/mL) activates a myeloblastoma oncoprotein (MYB) promoter (-687-+204)-driven luciferase reporter in K562 cells. (B, C) Clofazimine (CFZ) (5 μM) rapidly reduces p65 protein level (B) but does not alter p65 mRNA (C) in K562 cells. (D) CFZ (5 μM, 24 h) reduces a nuclear factor kappa B response element-driven reporter in K562 cells. (E) MG132 or lactacystin (10 μM, 6 h pretreatment) prevents the CFZ (5 μM, 1 h)-mediated reduction in p65 protein level in K562 cells. (F) CFZ (5 μM, 1 h) induces ubiquitination of p65. Proteins were immunoprecipitated with a rabbit p65 antibody followed by western blotting with mouse ubiquitin or p65 anti- bodies. To avoid the possibility of detection of IgG heavy chain (~50 kDa), an IgG light chain-specific secondary antibody was used. (G) CFZ (5 μM, 24 h) reduces p65 protein in imatinib-resistant chronic phase chronic myeloid leukemia (CP-CML) cells. (H-K; treatment concentration and duration same as in Figure 3A-D) p65 over- expression in K562 cells mitigates CFZ-induced downregulation of MYB and peroxiredoxin 1 and upregulation of caspase 3 cleavage (H), CFZ-mediated apoptosis (I; dot plots in Online Supplementary Figure S9A), CD61 expression (J; dot plots in Online Supplementary Figure S9B) and generation of cellular reactive oxygen species (K). (L) p65 expression in CP-CML cells. Graphs (except L) illustrate the mean ± standard error of mean of three independent experiments. (B,E,H) are one represen- tative of three independent experiments. (F) is one representative of two independent experiments. *P<0.05, **P<0.01, ***P<0.001. (A) One-way analysis of vari- ance (ANOVA), (I-K) Two-way ANOVA followed by the Bonferroni post-test. (C, L); Kruskal-Wallis test followed by the Dunn test. (D, G) Unpaired two-tailed Student t-test. MYB: myeloblastoma oncoprotein; V: vehicle; NFκB: uclear factor kappa B; TNF-α: tumor necrosis factor-alpha; Lacta: lactacystin; IP: immunoprecipitation; WB: west- ern blot; UB: ubiquitin; PRDX1: peroxiredoxin 1; CC; cleaved caspase; DCFDA: 2',7'-dichlorofluorescein diacetate; GAPDH: glyceraldehyde 3-phosphate dehydroge- nase.
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